Genomic integrity is persistently threatened by endogenous damage from the cellular metabolism and exogenous environmental sources including UV, ionizing radiation, and mutagenic chemicals. To counter this damage, cells have evolved complex and interconnected DNA repair pathways to coordinate lesion detection and removal with other vital cellular processes such as DNA replication, transcription and recombination. Failure in any link can lead to genomic instability, a known precursor to both cancer and aging. The broad focus of this proposal is to understand the biological significance of the interaction between three proteins involved in preventing genomic instability: XPG, and the human RecQ family members BLM and WRN. XPG has important non-enzymatic functions in both transcription-coupled repair and in base-excision repair (BER) of oxidative damage, and mutations that block functions of XPG lead to a severe neurological and developmental disorder with symptoms of premature aging, Cockayne syndrome (XP-G/CS). Further, WRN and BLM both play important roles in the maintenance of genomic integrity during replication. The loss of BLM leads to Bloom's syndrome, characterized by a high incidence of cancer, and the loss of WRN leads to Werner's syndrome, a premature aging disorder with increased cancer incidence. Preliminary data suggests a novel replication-associated role for XPG. The hypotheses to be tested are that XPG functions with WRN or BLM in homologous recombination repair (HRR) of stalled or collapsed replication forks after DNA damage, and/or during normal telomere replication. Aim 1 will use model DNA substrates of HRR intermediates to investigate the mechanisms by which XPG affects the function of BLM and WRN enzymatic activities. Aim 2 will use immunofluorescence, cell fractionation, and immunoprecipitations to test the hypothesis that XPG localizes to sites of stalled or collapsed replication forks with WRN or BLM. Aim 3 will examine primary XP-G/CS cells for telomere integrity, and determine the cell cycle and DNA damage-induced conditions under which XPG localizes to the telomeres. These studies will provide valuable insights into the roles of highly multifunctional proteins to regulate crosstalk between DNA replication and repair. Both of these DNA transactions are critically important in the maintenance of genomic stability and defense against cancer and aging.